Binding of TMPRSS2-ERG to BAF Chromatin Remodeling Complexes Mediates Prostate Oncogenesis.

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.

"Stromal cells in prostate cancer pathobiology: friends or foes?"

The genomic, epigenetic and metabolic determinants of prostate cancer pathobiology have been extensively studied in epithelial cancer cells. However, malignant cells constantly interact with the surrounding environment-the so-called tumour microenvironment (TME)-which may influence tumour cells to proliferate and invade or to starve and die. In that regard, stromal cells-including fibroblasts, smooth muscle cells and vasculature-associated cells-constitute an essential fraction of the prostate cancer TME. However, they have been largely overlooked compared to other cell types (i.e. immune cells). Indeed, their importance in prostate physiology starts at organogenesis, as the soon-to-be prostate stroma determines embryonal epithelial cells to commit toward prostatic differentiation. Later in life, the appearance of a reactive stroma is linked to the malignant transformation of epithelial cells and cancer progression. In this Review, we discuss the main mesenchymal cell populations of the prostate stroma, highlighting their dynamic role in the transition of the healthy prostate epithelium to cancer. A thorough understanding of those populations, their phenotypes and their transcriptional programs may improve our understanding of prostate cancer pathobiology and may help to exploit prostate stroma as a biomarker of patient stratification and as a therapeutic target.

Genetic ablation of FASN attenuates the invasive potential of prostate cancer driven by Pten loss.

Loss of the tumor suppressor gene Pten in murine prostate recapitulates human carcinogenesis and causes stromal proliferation surrounding murine prostate intraepithelial neoplasia (mPIN), which is reactive to microinvasion. In turn, invasion has been shown to be regulated in part by de novo fatty acid synthesis in prostate cancer. We therefore investigated the effects of genetic ablation of Fasn on invasive potential in prostate-specific Pten knockout mice. Combined genetic ablation of Fasn and Pten reduced the weight and volume of all the prostate lobes when compared to single knockouts. The stromal reaction to microinvasion and the cell proliferation that typically occurs in Pten knockout were largely abolished by Fasn knockout. To verify that Fasn knockout indeed results in decreased invasive potential, we show that genetic ablation and pharmacologic inhibition of FASN in prostate cancer cells significantly inhibit cellular motility and invasion. Finally, combined loss of PTEN with FASN overexpression was associated with lethality as assessed in 660 prostate cancer patients with 14.2 years of median follow-up. Taken together, these findings show that de novo lipogenesis contributes to the aggressive phenotype induced by Pten loss in murine prostate and targeting Fasn may reduce the invasive potential of prostate cancer driven by Pten loss. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Blocking lipid synthesis induces DNA damage in prostate cancer and increases cell death caused by PARP inhibition.

Androgen deprivation therapy (ADT) is the primary treatment for prostate cancer; however, resistance to ADT invariably develops, leading to castration-resistant prostate cancer (CRPC). Prostate cancer progression is marked by increased de novo synthesis of fatty acids due to overexpression of fatty acid synthase (FASN), making this enzyme a therapeutic target for prostate cancer. Inhibition of FASN results in increased intracellular amounts of ceramides and sphingomyelin, leading to DNA damage through the formation of DNA double-strand breaks and cell death. We found that combining a FASNi with the poly-ADP ribose polymerase (PARP) inhibitor olaparib, which induces cell death by blocking DNA damage repair, resulted in a more pronounced reduction in cell growth than that caused by either drug alone. Human CRPC organoids treated with a combination of PARP and FASNi were smaller, had decreased cell proliferation, and showed increased apoptosis and necrosis. Together, these data indicate that targeting FASN increases the therapeutic efficacy of PARP inhibitors by impairing DNA damage repair, suggesting that combination therapies should be explored for CRPC.

Interplay of IGF1R and estrogen signaling regulates hematopoietic stem and progenitor cells.

Tissue stem cells often exhibit developmental stage-specific and sexually dimorphic properties, but the underlying mechanism remains largely elusive. By characterizing IGF1R signaling in hematopoietic cells, here we report that its disruption exerts sex-specific effects in adult hematopoietic stem and progenitor cells (HSPCs). Loss of IGF1R decreases the HSPC population in females but not in males, in part due to a reduction in HSPC proliferation induced by estrogen. In addition, the adult female microenvironment enhances engraftment of wild-type but not Igf1r-null HSPCs. In contrast, during gestation, when both female and male fetuses are exposed to placental estrogens, loss of IGF1R reduces the numbers of their fetal liver HSPCs regardless of sex. Collectively, these data support the interplay of IGF1R and estrogen pathways in HSPCs and suggest that the proliferation-promoting effect of estrogen on HSPCs is in part mediated via IGF1R signaling.

Adipose Triglyceride Lipase Is a Therapeutic Target in Advanced Prostate Cancer That Promotes Metabolic Plasticity.

Lipid metabolism plays a central role in prostate cancer. To date, the major focus has centered on de novo lipogenesis and lipid uptake in prostate cancer, but inhibitors of these processes have not benefited patients. A better understanding of how cancer cells access lipids once they are created or taken up and stored could uncover more effective strategies to perturb lipid metabolism and treat patients. Here, we identified that expression of adipose triglyceride lipase (ATGL), an enzyme that controls lipid droplet homeostasis and a previously suspected tumor suppressor, correlates with worse overall survival in men with advanced, castration-resistant prostate cancer (CRPC). Molecular, genetic, or pharmacologic inhibition of ATGL impaired human and murine prostate cancer growth in vivo and in cell culture or organoids under conditions mimicking the tumor microenvironment. Mass spectrometry imaging demonstrated that ATGL profoundly regulates lipid metabolism in vivo, remodeling membrane composition. ATGL inhibition induced metabolic plasticity, causing a glycolytic shift that could be exploited therapeutically by cotargeting both metabolic pathways. Patient-derived phosphoproteomics identified ATGL serine 404 as a target of CAMKK2-AMPK signaling in CRPC cells. Mutation of serine 404 did not alter the lipolytic activity of ATGL but did decrease CRPC growth, migration, and invasion, indicating that noncanonical ATGL activity also contributes to disease progression. Unbiased immunoprecipitation/mass spectrometry suggested that mutation of serine 404 not only disrupts existing ATGL protein interactions but also leads to new protein-protein interactions. Together, these data nominate ATGL as a therapeutic target for CRPC and provide insights for future drug development and combination therapies. SIGNIFICANCE: ATGL promotes prostate cancer metabolic plasticity and progression through both lipase-dependent and lipase-independent activity, informing strategies to target ATGL and lipid metabolism for cancer treatment.

Distinct mesenchymal cell states mediate prostate cancer progression.

In the complex tumor microenvironment (TME), mesenchymal cells are key players, yet their specific roles in prostate cancer (PCa) progression remain to be fully deciphered. This study employs single-cell RNA sequencing to delineate molecular changes in tumor stroma that influence PCa progression and metastasis. Analyzing mesenchymal cells from four genetically engineered mouse models (GEMMs) and correlating these findings with human tumors, we identify eight stromal cell populations with distinct transcriptional identities consistent across both species. Notably, stromal signatures in advanced mouse disease reflect those in human bone metastases, highlighting periostin's role in invasion and differentiation. From these insights, we derive a gene signature that predicts metastatic progression in localized disease beyond traditional Gleason scores. Our results illuminate the critical influence of stromal dynamics on PCa progression, suggesting new prognostic tools and therapeutic targets.

Therapeutic blocking of VEGF binding to neuropilin-2 diminishes PD-L1 expression to activate antitumor immunity in prostate cancer.

Prostate cancers are largely unresponsive to immune checkpoint inhibitors (ICIs), and there is strong evidence that programmed death-ligand 1 (PD-L1) expression itself must be inhibited to activate antitumor immunity. Here, we report that neuropilin-2 (NRP2), which functions as a vascular endothelial growth factor (VEGF) receptor on tumor cells, is an attractive target to activate antitumor immunity in prostate cancer because VEGF-NRP2 signaling sustains PD-L1 expression. NRP2 depletion increased T cell activation in vitro. In a syngeneic model of prostate cancer that is resistant to ICI, inhibition of the binding of VEGF to NRP2 using a mouse-specific anti-NRP2 monoclonal antibody (mAb) resulted in necrosis and tumor regression compared with both an anti-PD-L1 mAb and control immunoglobulin G. This therapy also decreased tumor PD-L1 expression and increased immune cell infiltration. We observed that the NRP2, VEGFA, and VEGFC genes are amplified in metastatic castration-resistant and neuroendocrine prostate cancer. We also found that individuals with NRP2High PD-L1High metastatic tumors had lower androgen receptor expression and higher neuroendocrine prostate cancer scores than other individuals with prostate cancer. In organoids derived from patients with neuroendocrine prostate cancer, therapeutic inhibition of VEGF binding to NRP2 using a high-affinity humanized mAb suitable for clinical use also diminished PD-L1 expression and caused a substantial increase in immune-mediated tumor cell killing, consistent with the animal studies. These findings provide justification for the initiation of clinical trials using this function-blocking NRP2 mAb in prostate cancer, especially for patients with aggressive disease.

Distinct mesenchymal cell states mediate prostate cancer progression.

Alterations in tumor stroma influence prostate cancer progression and metastatic potential. However, the molecular underpinnings of this stromal-epithelial crosstalk are largely unknown. Here, we compare mesenchymal cells from four genetically engineered mouse models (GEMMs) of prostate cancer representing different stages of the disease to their wild-type (WT) counterparts by single-cell RNA sequencing (scRNA-seq) and, ultimately, to human tumors with comparable genotypes. We identified 8 transcriptionally and functionally distinct stromal populations responsible for common and GEMM-specific transcriptional programs. We show that stromal responses are conserved in mouse models and human prostate cancers with the same genomic alterations. We noted striking similarities between the transcriptional profiles of the stroma of murine models of advanced disease and those of of human prostate cancer bone metastases. These profiles were then used to build a robust gene signature that can predict metastatic progression in prostate cancer patients with localized disease and is also associated with progression-free survival independent of Gleason score. Taken together, this offers new evidence that stromal microenvironment mediates prostate cancer progression, further identifying tissue-based biomarkers and potential therapeutic targets of aggressive and metastatic disease.

Circulating Cell-Free DNA in Renal Cell Carcinoma: The New Era of Precision Medicine.

Tumor biopsy is still the gold standard for diagnosing and prognosis renal cell carcinoma (RCC). However, its invasiveness, costs, and inability to accurately picture tumor heterogeneity represent major limitations to this procedure. Analysis of circulating cell-free DNA (cfDNA) is a non-invasive cost-effective technique that has the potential to ease cancer detection and prognosis. In particular, a growing body of evidence suggests that cfDNA could be a complementary tool to identify and prognosticate RCC while providing contemporary mutational profiling of the tumor. Further, recent research highlighted the role of cfDNA methylation profiling as a novel method for cancer detection and tissue-origin identification. This review synthesizes current knowledge on the diagnostic, prognostic, and predictive applications of cfDNA in RCC, with a specific focus on the potential role of cell-free methylated DNA (cfMeDNA).

Temporal evolution of cellular heterogeneity during the progression to advanced AR-negative prostate cancer.

Despite advances in the development of highly effective androgen receptor (AR)-directed therapies for the treatment of men with advanced prostate cancer, acquired resistance to such therapies frequently ensues. A significant subset of patients with resistant disease develop AR-negative tumors that lose their luminal identity and display neuroendocrine features (neuroendocrine prostate cancer (NEPC)). The cellular heterogeneity and the molecular evolution during the progression from AR-positive adenocarcinoma to AR-negative NEPC has yet to be characterized. Utilizing a new genetically engineered mouse model, we have characterized the synergy between Rb1 loss and MYCN (encodes N-Myc) overexpression which results in the formation of AR-negative, poorly differentiated tumors with high metastatic potential. Single-cell-based approaches revealed striking temporal changes to the transcriptome and chromatin accessibility which have identified the emergence of distinct cell populations, marked by differential expression of Ascl1 and Pou2f3, during the transition to NEPC. Moreover, global DNA methylation and the N-Myc cistrome are redirected following Rb1 loss. Altogether, our data provide insight into the progression of prostate adenocarcinoma to NEPC.

CREB5 Promotes Resistance to Androgen-Receptor Antagonists and Androgen Deprivation in Prostate Cancer.

Androgen-receptor (AR) inhibitors, including enzalutamide, are used for treatment of all metastatic castration-resistant prostate cancers (mCRPCs). However, some patients develop resistance or never respond. We find that the transcription factor CREB5 confers enzalutamide resistance in an open reading frame (ORF) expression screen and in tumor xenografts. CREB5 overexpression is essential for an enzalutamide-resistant patient-derived organoid. In AR-expressing prostate cancer cells, CREB5 interactions enhance AR activity at a subset of promoters and enhancers upon enzalutamide treatment, including MYC and genes involved in the cell cycle. In mCRPC, we found recurrent amplification and overexpression of CREB5. Our observations identify CREB5 as one mechanism that drives resistance to AR antagonists in prostate cancers.

Protocols for Studies on TMPRSS2/ERG in Prostate Cancer.

TMPRSS2/ERG is the most common type of gene fusions found in human prostate cancer. There are two important features of TMPRSS2/ERG fusions. One is that these gene fusions lead to ectopic expression of ERG, an ETS family transcription factor, in prostate epithelial cells from the 5' control region of an androgen/estrogen dual-responsive gene, TMPRSS2; the other is that ~60% of these fusions are generated via intrachromosomal deletion of the interstitial region between TMPRSS2 and ERG. To recapitulate these important aspects of TMPRSS2/ERG fusions, we generated several TMPRSS2/ERG knockin mouse models based on the endogenous Tmprss2 locus. We found that TMPRSS2/ERG represents an early event in prostate tumorigenesis, by sensitizing prostate cells for cooperation with other oncogenic events, such as PTEN-deficiency. We also found that the interstitial region between TMPRSS2 and ERG harbors at least one prostate tumor suppressor, ETS2, whose loss contributes to prostate cancer progression. In this protocol, we describe how these knockin mouse models can be utilized to study roles of TMPRSS2/ERG fusions in prostate cancer development both in vivo and in vitro.

LONG-NONCODING RNAs in gastroesophageal cancers.

Despite continuing improvements in multimodal therapies, gastro-esophageal malignances remain widely prevalent in the population and is characterized by poor overall and disease-free survival rates. Due to the lack of understanding about the pathogenesis and absence of reliable markers, gastro-esophageal cancers are associated with delayed diagnosis. The increasing understanding about cancer's molecular landscape in the recent years, offers the possibility of identifying 'targetable' molecular events and in particular facilitates novel treatment strategies and development of biomarkers for early stage diagnosis. At least 98% of our genome is actively transcribed into non-coding RNAs encompassing long non-coding RNAs (lncRNAs) constituted of transcripts longer than 200 nucleotides with no protein-coding capacity. Many studies have demonstrated that lncRNAs are functional genomic elements playing pivotal roles in main oncogenic processes. LncRNA can act at multiple levels developing a complex molecular network that can modulate directly or indirectly the expression of genes involved in tumorigenesis. In this review, we focus on lncRNAs as emerging players in gastro-esophageal carcinogenesis and critically assess their potential as reliable noninvasive biomarkers and in next generation targeted therapies.

Single-Cell Analysis Identifies LY6D as a Marker Linking Castration-Resistant Prostate Luminal Cells to Prostate Progenitors and Cancer.

The exact identity of castrate-resistant (CR) cells and their relation to CR prostate cancer (CRPC) is unresolved. We use single-cell gene profiling to analyze the molecular heterogeneity in basal and luminal compartments. Within the luminal compartment, we identify a subset of cells intrinsically resistant to castration with a bi-lineage gene expression pattern. We discover LY6D as a marker of CR prostate progenitors with multipotent differentiation and enriched organoid-forming capacity. Lineage tracing further reveals that LY6D+ CR luminal cells can produce LY6D- luminal cells. In contrast, in luminal cells lacking PTEN, LY6D+ cells predominantly give rise to LY6D+ tumor cells, contributing to high-grade PIN lesions. Gene expression analyses in patients' biopsies indicate that LY6D expression correlates with early disease progression, including progression to CRPC. Our studies thus identify a subpopulation of luminal progenitors characterized by LY6D expression and intrinsic castration resistance. LY6D may serve as a prognostic maker for advanced prostate cancer.

A Tale of Two Signals: AR and WNT in Development and Tumorigenesis of Prostate and Mammary Gland.

Prostate cancer (PCa) is one of the most common cancers and among the leading causes of cancer deaths for men in industrialized countries. It has long been recognized that the prostate is an androgen-dependent organ and PCa is an androgen-dependent disease. Androgen action is mediated by the androgen receptor (AR). Androgen deprivation therapy (ADT) is the standard treatment for metastatic PCa. However, almost all advanced PCa cases progress to castration-resistant prostate cancer (CRPC) after a period of ADT. A variety of mechanisms of progression from androgen-dependent PCa to CRPC under ADT have been postulated, but it remains largely unclear as to when and how castration resistance arises within prostate tumors. In addition, AR signaling may be modulated by extracellular factors among which are the cysteine-rich glycoproteins WNTs. The WNTs are capable of signaling through several pathways, the best-characterized being the canonical WNT/ß-catenin/TCF-mediated canonical pathway. Recent studies from sequencing PCa genomes revealed that CRPC cells frequently harbor mutations in major components of the WNT/ß-catenin pathway. Moreover, the finding of an interaction between ß-catenin and AR suggests a possible mechanism of cross talk between WNT and androgen/AR signaling pathways. In this review, we discuss the current knowledge of both AR and WNT pathways in prostate development and tumorigenesis, and their interaction during development of CRPC. We also review the possible therapeutic application of drugs that target both AR and WNT/ß-catenin pathways. Finally, we extend our review of AR and WNT signaling to the mammary gland system and breast cancer. We highlight that the role of AR signaling and its interaction with WNT signaling in these two hormone-related cancer types are highly context-dependent.

Gab1 and Mapk Signaling Are Essential in the Hair Cycle and Hair Follicle Stem Cell Quiescence.

Gab1 is a scaffold protein that acts downstream of receptor tyrosine kinases. Here, we produced conditional Gab1 mutant mice (by K14- and Krox20-cre) and show that Gab1 mediates crucial signals in the control of both the hair cycle and the self-renewal of hair follicle stem cells. Remarkably, mutant hair follicles do not enter catagen, the destructive phase of the hair cycle. Instead, hair follicle stem cells lose quiescence and become exhausted, and thus no stem cell niches are established in the bulges. Moreover, conditional sustained activation of Mapk signaling by expression of a gain-of-function Mek1(DD) allele (by Krox20-cre) rescues hair cycle deficits and restores quiescence of the stem cells. Our data thus demonstrate an essential role of Gab1 downstream of receptor tyrosine kinases and upstream of Shp2 and Mapk in the regulation of the hair cycle and the self-renewal of hair follicle stem cells.

MDM2 promotes cell motility and invasiveness through a RING-finger independent mechanism.

Recent studies connect MDM2 with increased cell motility, invasion and/or metastasis proposing an MDM2-mediated ubiquitylation-dependent mechanism. Interestingly, in renal cell carcinoma (RCC) p53/MDM2 co-expression is associated with reduced survival which is independently linked with metastasis. We therefore investigated whether expression of p53 and/or MDM2 promotes aggressive cell phenotypes. Our data demonstrate that MDM2 promotes increased motility and invasiveness in RCC cells (N.B. similar results are obtained in non-RCC cells). This study shows for the first time both that endogenous MDM2 significantly contributes to cell motility and that this does not depend upon the MDM2 RING-finger, i.e. is independent of ubiquitylation (and NEDDylation). Our data suggest that protein-protein interactions provide a likely mechanistic basis for MDM2-promoted motility which may constitute future therapeutic targets.